Mesothelioma response to carbon nanotubes is associated with an early and selective accumulation of immunosuppressive monocytic cells

BACKGROUND: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells. METHODS: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells. RESULTS: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development. CONCLUSIONS: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer

Lung accumulation of Th 17 cells but not T regs is limited in the absence of MyD88 signalling.

<p>Lymphocyte numbers in the bronchoalveolar lavage (BAL) fluid (<b>A</b>) and CD4+ T lymphocyte numbers in lung suspensions (<b>B</b>) obtained from wild-type (WT) and MyD88-knockout (MyD88-KO) mice 60 days after silica (SiO₂) or saline solution (NaCl) treatment. Expression of Foxp3 (<b>C</b> and <b>D</b>), IL-17A (<b>E</b>) and RORγ (<b>F</b>) analyzed by RTqPCR in lungs (<b>C</b>) and in lung CD4+ T lymphocytes (<b>D–F</b>) obtained from WT and MyD88-KO mice 60 days after silica or saline treatment. Expression of Foxp3 and IL-17A was normalized on that of 18S RNA. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a t-test. ns indicates no statistically significant difference and * = p<0.05 and ** = p<0.01 indicates statistically significant difference between values measured in silica-treated WT mice and silica-treated KO mice.</p

Lung inflammation but not collagen accumulation is reduced in MyD88-KO mice treated with silica particles.

<p>Neutrophil (<b>A</b>) and macrophage (<b>B</b>) numbers in the bronchoalveolar lavage (BAL) fluid of wild-type (WT) and MyD88-knockout (MyD88-KO) mice treated with silica (SiO₂, 2.5 mg/mouse) or saline solution (NaCl) and sacrificed at day 60. Quantification of IL-1β (<b>C</b>) by ELISA in the BAL fluid of WT and MyD88-KO mice treated with SiO₂ or NaCl at day 60. Lung fibrosis assessed by OH-proline contents (<b>D</b>) in WT and MyD88-KO mice after silica or saline instillation (d60). Bars represent means ± SEM (n = 4–6). These results were treated statistically by a t- test. ns indicates no statistically significant difference and ** = p<0.01 indicate statistically significant difference between values measured in silica-treated WT and silica-treated KO mice.</p

Pulmonary accumulation of T regs and Th17 lymphocytes is associated with the development of silica-induced lung fibrosis.

<p>Number of CD4+ (<b>A</b>) and CD8+ (<b>B</b>) analyzed by flow cytometry in lung cell suspensions obtained from wild-type mice after silica (SiO₂, d3 to d60) or saline (d0) instillation. Expression of Foxp3 (<b>C</b>) and IL-17A (<b>D</b>) analyzed by RTqPCR in the lungs obtained from wild-type mice after silica (d3 to d60) or saline (d0) treatment. Expression of Foxp3 and IL-17A was normalized on that of 18S RNA. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a Student Neuman-Keul's test. * = p<0.05, ** = p<0.01, *** = p<0.001 indicate statistically significant difference between values measured in saline-treated mice and silica-treated mice.</p

MyD88-KO mice treated with silica particles developed limited granuloma and neutrophil infiltration but marked fibrosis and lymphocyte accumulation.

<p>5-µm sections of paraffin-embedded lung tissue of wild-type (WT) and MyD88- knockout (MyD88-KO) mice treated with silica (SiO₂, day 60) were stained with hematoxylin and eosin (panels <b>A–B</b> and <b>I–J</b>; neutrophils are indicated by black arrows and lymphocyte aggregates by an asterisk), with Masson's trichrome (panels <b>C–F</b>) or with silver (panels <b>G–H</b>). Sections are representative of 3–4 mice examined. Magnification 50X (panels A and B), 100X (panels <b>C–H</b>) and 400X (panels I–J). Histological quantification of fibrotic nodule, diffuse fibrosis and lymphoid aggregate numbers (<b>K</b>). These results were treated statistically by a t- test. * = p<0.05, ** = p<0.01 indicate statistically significant difference between values measured in silica-treated WT and silica-treated KO mice.</p

Lung expression of TGF-β1, IL-10 and PDGF-B is not reduced in MyD88-KO mice treated with silica particles.

<p>Lung expression of TGF-β1 (<b>A</b>), IL-10 (<b>B</b>) and PDGF-BB (<b>C</b>) analyzed by RTqPCR in tissue or by ELISA in BAL of wild-type (WT) and MyD88-knockout (MyD88-KO) mice 60 days after silica (SiO₂) or saline (NaCl) treatment. Expression of PDGF B (<b>D</b>) analyzed by RTqPCR in lung CD4+ T lymphocytes purified from NaCl- or SiO₂-treated WT and MyD88-KO mice at day 60. Expression of TGF-β1, IL-10 and PDGF-B was normalized on that of 18S RNA. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a t- test and no statistically significant difference (ns) between values measured in silica-treated WT mice and silica-treated KO mice was noted.</p

CCR2+ monocytic myeloid-derived suppressor cells (M-MDSCs) inhibit collagen degradation and promote lung fibrosis by producing transforming growth factor-β1.

Monocytes infiltrating scar tissue are predominantly viewed as progenitor cells. Here, we show that tissue CCR2+ monocytes have specific immunosuppressive and profibrotic functions. CCR2+ monocytic cells are acutely recruited to the lung before the onset of silica-induced fibrosis in mice. These tissue monocytes are defined as monocytic myeloid-derived suppressor cells (M-MDSCs) because they significantly suppress T-lymphocyte proliferation in vitro. M-MDSCs collected from silica-treated mice also express transforming growth factor (TGF)-β1, which stimulates lung fibroblasts to release tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of metalloproteinase collagenolytic activity. By using LysMCreCCR2loxP/loxP mice, we show that limiting CCR2+ M-MDSC accumulation reduces the pulmonary contents of TGF-β1, TIMP-1 and collagen after silica treatment. M-MDSCs do not differentiate into lung macrophages, granulocytes or fibrocytes during pulmonary fibrogenesis. Collectively, our data indicate that M-MDSCs contribute to lung fibrosis by specifically promoting a non-degrading collagen microenvironment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Robust fibrotic response in the absence of innate immune receptors or ligands.

<p>Hydroxyproline (OH-proline) content assessed in lung homogenates of mice 60 days after saline (NaCl) or silica (SiO₂) instillation in wild-type (WT), IL-1R- (<b>A</b>), ASC- (<b>B</b>), NALP3- (<b>C</b>), IL-1β- (<b>D</b>), TRIF- (<b>E</b>), TLR2/4- (<b>F</b>), TLR3- (<b>G</b>), γδTCR- (<b>H</b>), IL-23p19- (<b>I</b>) knockout (KO) mice. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a t- test. ns indicates no statistically significant difference and ** = p<0.01 indicates statistically significant difference between values measured in silica-treated WT and silica-treated KO mice.</p

Author Correction: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization

International audienceBackground: Inflammasome-activated IL-1β plays a major role in lung neutrophilic inflammation induced by inhaled silica. However, the exact mechanisms that contribute to the initial production of precursor IL-1β (pro-IL-1β) are still unclear. Here, we assessed the implication of alarmins (IL-1α, IL-33 and HMGB1) in the lung response to silica particles and found that IL-1α is a master cytokine that regulates IL-1β expression. Methods: Pro-and mature IL-1β as well as alarmins were assessed by ELISA, Western Blot or qRT-PCR in macrophage cultures and in mouse lung following nano-and micrometric silica exposure. Implication of these immune mediators in the establishment of lung inflammatory responses to silica was investigated in knockout mice or after antibody blockade by evaluating pulmonary neutrophil counts, CXCR2 expression and degree of histological injury. Results: We found that the early release of IL-1α and IL-33, but not HMGB1 in alveolar space preceded the lung expression of pro-IL-1β and neutrophilic inflammation in silica-treated mice. In vitro, the production of pro-IL-1β by alveolar macrophages was significantly induced by recombinant IL-1α but not by IL-33. Neutralization or deletion of IL-1α reduced IL-1β production and neutrophil accumulation after silica in mice. Finally, IL-1α released by J774 macrophages after in vitro exposure to a range of micro-and nanoparticles of silica was correlated with the degree of lung inflammation induced in vivo by these particles. Conclusions: We demonstrated that in response to silica exposure, IL-1α is rapidly released from pre-existing stocks in alveolar macrophages and promotes subsequent lung inflammation through the stimulation of IL-1β production. Moreover, we demonstrated that in vitro IL-1α release from macrophages can be used to predict the acute inflammogenic activity of silica micro-and nanoparticles